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EFFECTIVENESS OF CRUDE EXTRACT OF MAYANA LEAVES FOR BLOOD COAGULATION PROCESS
An Undergraduate ThesisPresented to the Faculty of the
College of Allied Health SciencesMedical Technology Department
Cagayan State UniversityAndrew’s Campus, Tuguegarao City
In Partial Fulfillment Of the Requirements for the Degree
Bachelor of Science in Medical Technology
ByRhoe Anne C. CalabazaronRandolph Ryan G. Gasmin
Abigail C. GuinucayJocelyn P. Oledan
Niki Valencia D. PamittanRachelle I. Tugad
March 2012
Chapter I
THE PROBLEM AND ITS BACKGROUND
Introduction
Use of herbs and medicinal plants as the first medicines is a universal phenomenon.
Every culture on Earth, through written or oral tradition, has relied on the vast variety of
healing plants for their therapeutic properties. The majority of herbal products available
today originated from the same traditional formulas or ingredients. The use of herbal
medicines predates human history. Herbal medicine creates a more potent, effective and
efficient treatment to ensure quicker responses to re-establish health and balance.
Most people used herbal medicines to treat their wounds, especially in rural areas.
Some people use herbal perching for the leaves to produce extract and put the leaves on
the wound. They believed that herbal plants are effective in treating wounds especially in
minimizing its bleeding. Not knowing that these herbal plants are also effective in
minimizing the bleeds on wounds which can be used as an immediate treatment for
hemostasis. Herbal medicine can provide a better quality of life
(http://arightpath.com/herbal-medicine/importance.html).
In rural areas, people resorted in using herbal medicine to treat their wound without
seeking consultation from a clinic or a hospital. They just get herbal medicine from their
backyard because it’s available locally. Nowadays, people are educated about the
usefulness of herbal medicines. All parts of a medicinal plant are used: roots, branches,
twigs, stems, leaves, flowers, fruit and seeds. All have separate functions; for instance,
the leaves are the most superficial part of a plant and therefore are potent at treating acute
and superficial diseases.
Many people believe that herbs are just as effective as drugs, but without the side
effects. Herbs do perform many healing functions in the body, but they must be used
appropriately and recommended by a trained professional. Although herbal remedies are
less likely than most conventional medicines to cause side effects, herbs nevertheless can
be very potent.
However, the use of locally available medical plants has been advocated by the
Department of Health. Many local plants and herbs in the Philippine backyard and field
have been found to be effective in the treatment of common ailments as attested to by the
National Science Development Board. The use of locally available medical plants has
been advocated by the Department of Health. Many local plants and herbs in the
Philippine backyard and field have been found to be effective in the treatment of common
ailments as attested to by the National Science Development Board. (Cuevas, 2007)
The folkloric used of Mayana are for carminatives, bruises and sprains, headaches,
mild bleeding of wounds, sinusitis, dyspepsia, and eye drops for eye irritations. The use
of mayana is reported as an Asian traditional medicine for asthma, angina, bronchitis,
epilepsy, insomnia, skin rashes (http://www.stuartxchange.com/ index.html).
Blood is of prime importance in the normal physiologic function of our major
organ systems. When you cut yourself, the process of coagulation begins by the
formation of a blood clot. This is followed shortly after by digestion or breakdown of the
clot. Patients clot and/or bleed because of a variety of identifiable hemostatic
abnormalities. The property of the circulation where the circulating fluid is maintained
within the blood vessels is referred to as hemostasis. Logical and effective treatment
depends upon the proper identification of the abnormality. (Hemostasis Basics, Dade
Behring, 2000).
This research entitled “Effectual Use of Crude Extracts of Mayana (Coeus blumei
benth) leaves for blood coagulation “attempts to determine the effective component of the
mayana leaves in blood clotting time. Furthermore, this study was conducted to test the
effectiveness of cephalin as a thromboplastic agent.
Theoretical Framework
Although the concept of the coagulation cascade represented a significant advance
in the understanding of coagulation and served for many years as a useful model, more
recent clinical and experimental observations demonstrate that the cascade/waterfall
hypothesis does not fully and completely reflect the events of hemostasis in vivo.
(Theories of Blood Coagulation; http://aphon.org/files/public/theories_of_coag.pdf)
The Classic Theory of Coagulation as Proposed by Paul Morawitz (1958), in which
the presence of calcium and thromboplastin, prothrombin was believed to be converted to
thrombin. In turn, the thrombin converted fibrinogen to fibrin, enabling the formation of a
fibrin clot. Morawitz posited that all the ingredients of clotting were present in circulating
blood and that the fact that such blood did not normally clot was due to the lack of a
wettable surface in the blood vessels.
Another related theory is the Coagulation Cascade Model by Macfarlane (1964)
appeared in the journal Nature described each clotting factor as a proenzyme that could
be converted to an active enzyme. The “cascade” and “waterfall” models suggested that
the clotting sequences were divided into 2 pathways. Coagulation could be initiated via
an “intrinsic pathway,” so named because all the components were present in blood, or by
an “extrinsic pathway,” in which the subendothelial cell membrane protein, tissue factor
(TF), was required in addition to circulating components. The initiation of either pathway
resulted in activation of FX and the eventual generation of a fibrin clot through a
common pathway (Luchtman-Jones & Broze, 1995).
Furthermore, a major development of Cell-Based Model of Coagulation was the
discovery that exposure of blood to cells that express TF on their surface is both
necessary and sufficient to initiate blood coagulation in vivo. This finding led to the
belief that the intrinsic pathway (the contact system) does not have a true physiological
role in hemostasis (Hoffbrand et al., 2005). Very recent evidence suggests that although
FXII deficiency does not result in bleeding problems, the absence of FXII does protect
against pathological thrombosis (Kleinschnitz et al., 2006).
In the theory of Herbalism, the herbs have been used since the dawn of time as
medicines and, in fact, many common drugs are in actual fact, made from herbal extracts.
The natural chemical properties of certain herbs have been shown to contain of
themselves, medicinal value. However, unlike conventional medicine, herbalists use the
'whole' herb or plant rather than isolating and breaking down chemical compounds and
then synthesising it. This is because the plant, being a part of Nature, is said to represent
perfect balance; healing requires the natural combination of elements in the plant or herb,
not just a single chemical within it.
In relation with the use of Mayana leaves in hemostasis, the crude extract of this
herb is also effective in blood clothing time. This is very useful in treating wounds. It can
also be used as first aid especially in case of immediate management to prevent blood
loss.
Research Paradigm
The Mayana and Cephalin are the Independent variables and the blood coagulation
is the dependent variable. But there is a possibility that biological factors such as
temperature, different blood types and their concentration as the intervening variables
might affect the blood coagulation process.
Figure 1
Independent Variables Intervening Variables Dependent Variables
Figure 1. Shows the Independent, Intervening and Dependent Variables on the Effectual Use of the Crude Extract of Mayana Leaves (Coeus Blumei benth) for Blood Coagulation Time in Different Blood Types.
Mayana (Coeus Blumei benth)
- Temperature- Blood type- pH
Blood Coagulation
Cephalin(Positive Control)
Statement of the Problem
The study is designed to find out the effectual use of mayana leaves extract for
blood coagulation.
Specifically, it answers the following questions:
1. What is the medicinal composition of mayana leaves that is contributory to blood
coagulation?
2. What is the extent of effectiveness of mayana leaves extract to hasten blood
coagulation time.
3. What health risk possibilities does mayana leaves extract have to human body
system?
4. Is there a significant difference in blood coagulation time between the four
different blood types using mayana leaves extract?
Assumptions
The assumptions of the study are presented in this from:
1.
Significance of the Study
The study is conducted to find out the effectiveness of mayana leaves extract as to
the blood coagulation. The results of the study are expected to benefit the following:
Remote areas. The researcher’s findings in this study would help the people inform in
remote areas where there is no access to hospitals or clinic that herbal plant is essential to
speed up the blood clot of the lesion. Furthermore, this study would provide additional
option aside from the cephaline or any other commercially available medicine for wound
in cases of emergency situations.
Doctors. The results of this research will aid the doctors to advise what kind of herbal
plant is efficient in treating an abrasion in case of urgent situation.
Public health nurse. The findings of this research will endow additional knowledge
regarding health information given or trained by public health nurse about the
effectiveness of mayana leaves extract to the blood coagulation process.
Researchers. The study would also serve as a proposal and a guide to the future
researchers on what variables they would embrace or focus as they carry out another
study associated to this subject matter.
Scope and Limitation of the Study
The scope of the study is on the effectiveness of crude extract of mayana leaves in
aiding the blood coagulation process. It will be conducted at the laboratory of Cagayan
State University, College of Allied Health Science. The blood types to be used in this
study are decided to be blood type A, B, AB and O. This is to determine the significant
difference of the different blood types on their blood coagulation time when the crude
extract of mayana leaves is used in comparable with cephalin known for its
thromboplastic agent. Since there is some sort of sensitivity in this experimental study,
the accuracy of the result of experimentation varies on the capacity of the researchers,
condition of the blood samples and the materials and instruments that are available to use.
Definition of Terms
The following terms are defined to assist a clear understanding of the study:
Blood clotting. The conversion of fluid blood into a coagulum that involves shedding of
blood, release of thromboplastin, inactivation of heparin, conversion of prothrombin to
thrombin, interaction of thrombin with fibrinogen to form an insoluble fibrin network,
and contraction of the network to squeeze excess fluid.
Blood coagulation. A complex process by which blood forms clots.
Cephalin. A thromboplastic substance which initiates the process of blood coagulation.
Clotting factor. Any substance in the blood that is essential for blood to coagulate.
Crude Extracts. The liquid form of the mayana made by purging of the leaves.
Effectiveness. The fulfilling result of the herbal plants used in the clotting time.
Fibrin. A fibrous, non-globular protein involved in the clotting of blood. It is a fibrillar
protein that is polymerized to form a "mesh" that forms haemostatic plug or clot over a
wound site.
Fibrinogen. A clotting factor and is required to prevent major blood loss.
Hemophilia. A rare bleeding disorder in which the blood doesn't clot normally.
Hemostatic disorder. Conditions that affect the ability of blood to clot properly.
Hemostasis. Entire mechanism by which bleeding from an injured blood vessel is
spontaneously controlled and stopped; Process by which spontaneous or induced
hemorrhage is stopped.
Herbal plant. Medicinal herbs to prevent and treat diseases and ailments or to promote
health and healing.
Mayana leaves. Commonly used as ornamental plant in the country due to its purplish
foliage, mayana can grow in the different kinds of habitat. It is one of the traditionally
used folklore medicine and it is primarily used for pain, sore, swelling and cuts and in
other instances as adjunct medication for delayed menstruation and diarrhea.
Prothrombin. A plasma protein that is converted to thrombin during blood clotting.
Thromboplastic agent. An agent causing or increasing blood-clot formation.
Thromboplastin. Enzyme in blood platelets; A blood-clotting factor in blood platelets
that converts prothrombin to thrombin.
Time. This word refers to the duration of time the blood will clot if the herbal plants are
used during bleeding.
Chapter II
REVIEW OF RELATED LITERATURE AND STUDIES
MAYANA
Mayana (Coeus blumei benth.) is an erect, branched, fleshy annual herb, about 1 m
high. The stems are purplish and four-angled. Leaves are blotched or colored, ovate, 5-10
cm long, with toothed margins. Flowers are purplish, numerous, in simple or branched
inflorescences, 15-30 cm long. It is one of the traditionally used folklore medicine and is
primarily used for pain, sore, swelling and cuts and in other instances as adjunct
medication for delayed menstruation and diarrhea. This plant can also make volatile oils
and cure bruises and sprains, carminative, headache, mild bleeding of wounds, sinusitis,
dyspersia and eye inflammation, (http://www.filipinoherbshealingwonders.filipino
vegetarianrecipe.com/mayana.htm).
According to Janzen (2005), Vachellia mayana probably represents a wet-forest
edition of V. cornigera. Undoubtedly, the two taxa are very closely related, having many
vegetative and floral characteristics in common. However, the large leaflets (more than
10 mm long), the rachis glands between each pinna pair, and the inflorescence, which
narrows toward the elongated and pointed apex separate this species from the closely
related V. cornigera and V. sphaerocephala. Also, the pair of blade-like longitudinal
flanges extending from the spine base to apex separates V. mayana from all other species
of ant-acacia. As is typical of most ant-acacias, none of the individuals of V. mayana
tested positive for cyanide production. Vachellia mayana is one of the rarer of the ant-
acacias. Collecting data from the few collections observed indicate that it has pinkish
flowers and varies in size from a shrub to a small tree to 10 m tall. Most collections
indicate that Vachellia mayana occurs as scattered individuals in moist lowland forests.
Janzen (2005) reported an individual from old second growth cornfield
regeneration where the forest was about 15 m tall. Unlike most wet forest ant-acacias,
Beltian body production in Vachellia mayana is extremely high. On developing leaves,
nearly all of the leaflets contain Beltian bodies, and these bodies are usually about 2 mm
long and up to 0.8 mm wide.
Mayana grows well in open areas with moist, well-drained and friable soil.
Occasionally cultivated throughout the Philippines. Common garden plant. It flowers all
year round. The plant is deeply rooted. Prefers warm and moist habitat, sensitive to
dryness. Soil should be well-drained, and rich in humus to produce higher yields. Use
seeds for propagation.
The mature fresh leaves of Mayana are harvested 2 to 3 months after planting.
Leaves are picked leaving the branches on the plant to allow it to flower and produce
seeds for the next season. The leaves are air-dried until they crumble when crushed with
the fingers. Store in amber colored bottles in a cool, dry place.
Chemists from the University of the Philippines isolated sterols and triterpenes
from the leaves of mayana and it exhibited analgesic, anti-inflammatory and
antimicrobial activities. Another interesting component of the plant is its high rosmarinic
acid content. This compound was noted for its high biological activities; prominent of
those are its anti-inflammatory and anti-oxidant properties. It may be grown in the garden
in bright, indirect light, or in practical shade, and will survive full sun exposure. It is
commonly used as ornamental plant in the country due to its purplish foliage; mayana can
grow in the different kinds of habitat. This traditional uses of mayana are scientifically
supported by studies here and abroad, (http://www.herbanext.com/herbs-mayana).
Cris (2008) conducted a study on the Feasibility of Using Mayana (Colleus
scutellarioides) as Biological Stain. Results showed that the extract of the mayana leaves
can be partly used as biological stain for plant cell. He concluded that almost all the cells
in samples can be seen the nucleus and the cell wall of the onion cell. In comparing the
mayana extract as biological stain from the commercial ones, many biological stain can’t
be an alternative for the commercial ones because the mayana extract can’t highlight
some parts of the plant cells. Cris’ study is unscientific because the explanation is
insufficient.
CEPHALIN
Phosphatidylethanolamine (cephalin, sometimes abbreviated PE) is a lipid found in
biological membranes. It is synthesized by the addition of CDP-ethanolamine to
diglyceride, releasing CMP. S-adenosyl methionine can subsequently methylate the
amine of phosphatidyl ethanolamine to yield phosphatidyl choline.
Cephalin is a phospholipid, which is a lipid derivative. It is not to be confused with
the molecule of the same name that is an alkaloid constituent of Ipecac. Cephalin is found
in all living cells, although in human physiology it is found particularly in nervous tissue
such as the white matter of brain, nerves, neural tissue, and in spinal cord. Whereas
lecithin is the principal phospholipid in animals, cephalin is the principal one in bacteria.
As a polar head group, phosphatidylethanolamine (PE) creates a more viscous lipid
membrane compared to phosphatidylcholine (PC). For example, the melting temperature
of di-oleoyl-PE is -16C while the melting temperature of di-oleoyl-PC is -20C. If the
lipids had two palmitoyl chains, PE would melt at 63C while PC would melt already at
41C. Lower melting temperatures correspond, in a simplistic view, to more fluid
membranes, (Wan et al. Biochemistry 47 2008).
A study of the activity of purified cephalin and lecithin as antigens in the
complement-fixation test and in the animal body is described. The cephalin and lecithin
used were both prepared from beef brain and the lecithin from beef liver and beef heart as
well. These phosphatides, dispersed by several methods, were inactive, outside the lytic
or anticomplementary range, in the in vitro test. Reinforcement with cholesterol had no
effect. The lecithins, when preserved under acetone to prevent oxidation, had neither lytic
nor anticomplementary properties.
The cephalins were all markedly anticomplementary; only very large amounts,
which were unsuitable for testing because of the opacity of the solutions, were lytic.
Immunization studies were carried out with three samples of purified cephalin, prepared
from brain tissue, and with two samples of purified lecithin, one from liver and one from
brain; also, for control, with commercial preparations of both cephalin and lecithin. The
sera of rabbits that received mixtures of the purified phosphatides and swine serum did
not fix complement with either the purified or the commercial phosphatides, or with beef-
heart extracts. Thus, there was no evidence of the antigenic action of these substances in
the tissues. The sera of rabbits inoculated with mixtures of the commercial phosphatides,
cephalin and lecithin, with swine serum fixed complement with the commercial
preparation used for inoculation, and with the Wassermann antigens but not with the
purified phosphatides.
This result strongly suggests that the specific activity of the commerical
preparations is associated with some contaminating substance rather than with lecithin or
cephalin itself. The advantage of a precise serologic method was demonstrated, since, by
its use, reactions which would otherwise have been regarded as significant were shown to
be non-specific.
Adsorption of commercial cephalin or lecithin on collodion particles did not confer
upon them any demonstrable immunizing properties; it did increase the
anticomplementary action of cephalin, (Related foreign study by Augustus Wadsworth et.
al. “A Study of the Antigenic Properties of Lecithin and Cephalin”).
Study has been made of the rat liver protein catalyzed exchange of both lecithin
and cephalin between liposomes and rat liver mitochondria. It has been shown that the
exchange activity of these two phospholipids by the protein is almost the same and is
apparently not dependent on the nature of donor liposomes. In contrast the spontaneous
exchange activity of the above phospholipids strictly depends on the type of donor
liposomes. Moreover, the spontaneous exchange of lecithin at any incubation time
appears to be almost 100% higher than that of cephalin, (Related foreign study by F M
Ruggiero et. al. “Comparative Study of Lecithin and Cephalin Exchange between
Liposomes and Mitochondria”).
In the original Hanger’s method of cephalin-cholesterol flocculation test (CCF
test), crude cephalin from sheep brain was used as a component of the CCF reagent, but
in Japan that from ox brain has also been utilized for the same purpose. Crude cephalins
from various sources were separate into five fractions by Folch in 1942, the first fraction
mainly consisting of inositol phospholoipid, the third of phosphatidyl serine and the fifth
of phosphatidyl ethanolamine, respectively. The second and forth fractions gave no
additional component other than those described above. Recently plasmalogens were also
reported by Klenk and others to be a component of the fifth fraction above mentioned.
In spite of many informations about the chemical nature of cephalin, it is not yet
certain whether a single component of cephalin fraction or some mixture of components
would participate in CCF reaction. On the other hand, CCF reagents available on the
market are known to differ widely in their flocculation activity according to their makers
and more or less with bathces of preparation. The inconstancy of reactivities among
batches of preparation may lead to confusion of clinical evaluation of CCF test followed
by loss of reliance on the test. These facts have hindered the CCF test from its broad
application in Japan. The differences in reactivities have not yet been chemically
explained. From the knowledge in reactivities have not yet been chemically explained.
From the knowledge about cephalin mixture, however, it is assumed that any single
fraction of cephalin may be the most potent in reactivity and others non-reactive or
inhibitory. The differences in reactivities of reagents above mentioned may be attributed
to the relative amount of the active component in cephalin preparation used in the test. If
a single component is reactive, it may be possible properly to control its reactivity by
means of admixture of a non-reactive or inhibitory. The differences in reactivities of
reagents above mentioned may be attributed to the relative amount of the active
component in cephalin preparation used in the test. If a single component is reactive, it
may be possible properly to control its reactivity by means of admixture of a non-reactive
or an antagonistical lipid matter in its purified state, (Related foreign study “Study on
Cephalin-Cholesterol-Flocculation Reaction).
Nine partial thromboplastin (cephalin) reagents have been compared in a parallel
investigation of groups of patients on 'long-term' anticoagulants, a group with moderate
haemophilia, and patients on heparin infusion. Results with the seven commercial
reagents and a human cephalin extract have been correlated with those of a specially
prepared and standardized reference preparation of human brain origin.
The comparison was similar in principle to that of the prothrombin time
thromboplastin standardization using the British Comparative Thromboplastin (BCT).
Results, which for comparative purposes were expressed as ratio of patients' cephalin
times to control cephalin times, varied greatly in all three groups. In the oral
anticoagulant group some of the commercial reagents were particularly insensitive to the
'intrinsic' clotting defect. The correlation between the 'standardized preparation' and the
other reagents was not good and the use of a reference cephalin material for quality
control of cephalin time tests does not appear promising.
In moderate haemophilia the commercial reagents were either relatively poor 'at
picking out the clotting defect compared with the 'standardized preparation' or gave such
a bad endpoint that the results were not dependable. The poor endpoint also limited the
dependability of the results of all but the 'standardized preparation' and two of the
commercial reagents in controlling heparin administration.
In view of these standardization difficulties, which cannot apparently be
resolved.by the use of reference material, there is need for bulk, routine supplies of a
sensitive, standardized cephalin reagent giving good reproducible endpoints. The method
for the provision of such material in a recently introduced national supply scheme is
described. The partial thromboplastin (cephalin) time is employed as an overall measure
of 'intrinsic' blood clotting. Its main applications are in the screening for hereditary and
acquired 'intrinsic' clotting defects and in assessing the intrinsic defect during oral
anticoagulant treatment. The test involves the recalcification of platelet-poor plasma in
the presence of a crude phospholipid extract. The origins of the latter vary and different
animal tissues are used in their preparation. In addition 'home-made' phospholipid
extracts from human brain may be made and used at individual hospitals.
The present report describes the results encountered in clinical practice when a
variety of reagents currently available in Britain are used. An attempt has been made to
correlate these reagents with a standardized cephalin preparation. The comparison of the
cephalin reagents has been on patients on long-term anticoagulants (nicoumalone
therapy), a group with moderately severe haemophilia, and patients on heparin therapy.
On the basis of the findings a system for standardizing the cephalin time test has been
introduced and involving the supply of a sensitive standardized cephalin preparation,
(Related foreign study by L. Poller et.al. “The Partial Thromboplastin (cephalin) Time
Test”).
BLOOD COAGULATION
Theories on the coagulation of blood have existed since antiquity. Physiologist
Johannes Müller (1801-1858) described fibrin, the substance of a thrombus. Its soluble
precursor, fibrinogen, was thus named by Rudolf Virchow (1821-1902), and isolated
chemically by Prosper Sylvain Denis (1799-1863). Alexander Schmidt suggested that the
conversion from fibrinogen to fibrin is the result of an enzymatic process, and labeled the
hypothetical enzyme thrombin and its precursor prothrombin. Arthus discovered in 1890
that calcium was essential in coagulation. Platelets were identified in 1865, and their
function was elucidated by Giulio Bizzozero in 1882(Fogoros, 2003). The theory that
thrombin is generated by the presence of tissue factor was consolidated by Paul Morawitz
in 1905. At this stage, it was known that thrombokinase/thromboplastin (factor III) is
released by damaged tissues, reacting with prothrombin (II), which, together with
calcium (IV), forms thrombin, which converts fibrinogen into fibrin (I).
Coagulation factors the remainder of the biochemical factors in the process of
coagulation were largely discovered in the 20th century. A first clue as to the actual
complexity of the system of coagulation was the discovery of proaccelerin (initially and
later called Factor V) by Paul Owren (1905-1990) in 1947. He also postulated its function
to be the generation of accelerin (Factor VI), which later turned out to be the activated
form of V (or Va); hence, VI is not now in active use. Factor VII (also known as serum
prothrombin conversion accelerator or proconvertin, precipitated by barium sulfate) was
discovered in a young female patient in 1949 and 1951 by different groups. Factor VIII
turned out to be deficient in the clinically recognized but etiologically elusive hemophilia
A; it was identified in the 1950s and is alternatively called antihemophilic globulin due to
its capability to correct hemophilia A.
Factor IX was discovered in 1952 in a young patient with hemophilia B named
Stephen Christmas (1947-1993). His deficiency was described by Dr. Rosemary Biggs
and Professor R.G. MacFarlane in Oxford, UK. The factor is, hence, called Christmas
Factor or Christmas Eve Factor. Christmas lived in Canada, and campaigned for blood
transfusion safety until succumbing to transfusion-related AIDS at age 46. An alternative
name for the factor is plasma thromboplastin component, given by an independent group
in California.
Hageman factor, now known as factor XII, was identified in 1955 in an
asymptomatic patient with a prolonged bleeding time named of John Hageman. Factor X,
or Stuart-Prower factor, followed, in 1956. This protein was identified in a Ms. Audrey
Prower of London, who had a lifelong bleeding tendency. In 1957, an American group
identified the same factor in a Mr. Rufus Stuart. Factors XI and XIII were identified in
1953 and 1961, respectively. The view that the coagulation process is a cascade or
waterfall was enunciated almost simultaneously by MacFarlane in the UK and by Davie
and Ratnoff in the USA, respectively (Hara, 2005).
BLOOD COAGULATION MECHANISM
Blood Clotting is one of the three mechanisms that reduce the loss of blood from
broken blood vessels. These three mechanisms are Vascular Spasm, platelet plug
formation and blood clotting. In vascular spasm the smooth muscle in blood vessel walls
contracts immediately the blood vessel is broken. This response reduces blood loss for
some time, while the other haemostatic mechanisms become active. The clotting
mechanism is one of the most important and complex of physiologic systems. Blood must
flow freely through the blood vessels in order to sustain life. But if a blood vessel is
traumatized, the blood must clot to prevent life from flowing away. Thus, the blood must
provide a system that can be activated instantaneously – and that can be contained locally
– to stop the flow of blood. This system is called the clotting mechanism (Fogoros, 2003).
When blood platelets encounter a damaged blood vessel, platelet plug formation
will occur to help close the gap in the broken blood vessel. The key stages of this process
are called platelet adhesion, platelet release reaction, and platelet aggregation. Following
damage to a blood vessel, vascular spasm occurs to reduce blood loss while other
mechanisms also take effect. Blood platelets congregate at the site of damage and a mass
to form a platelet plug. This is the beginning of the process of the blood breaking down
from its usual liquid form in such a way that its constituents play their own parts in
processes to minimize blood loss. Blood normally remains in its liquid state while it is
within the blood vessels but when it leaves them the blood may thicken and form a gel
(coagulation). Blood clotting technically blood coagulation is the process by which
(liquid) blood is transformed into a solid state. This blood clotting is a complex process
involving many clotting factors (incl. calcium ions, enzymes, platelets, damaged tissues)
activating each other. (Kozier, 2006)
The three stages of this process are Formation of Prothrombinase, prothrombin
converted into the enzyme thrombin, and fibrinogen (soluble) converted to fibrin
(insoluble). Prothrombinase which is the stage one can be formed in two ways, depending
of which of two systems or pathways apply. These are Intrinsic System and extrinsic
System. Intrinsic System is initiated by liquid blood making contact with a foreign
surface, i.e. something that is not part of the body. The Extrinsic System is initiated by
liquid blood making contact with damaged tissue. Both the intrinsic and the extrinsic
systems involve interactions between coagulation factors.
These coagulation factors have individual names but are often referred to by a
standardised set of Roman Numerals, e.g. Factor VIII (antihaemophilic factor), Factor IX
(Christmas factor). In stage two, Prothrombin converted into the enzyme Thrombin;
Prothrombinase formed in stage one converts prothrombin, which is a plasma protein that
is formed in the liver, into the enzyme thrombin; and in stage tree Fibrinogen (soluble)
will be converted to Fibrin (insoluble). In turn, thrombin converts fibrinogen (which is
also a plasma protein synthesized in the liver) into fibrin. Fibrin is insoluble and forms
the threads that bind the clot (Hara, 2005).
MEDICINAL PLANTS
In 2005, the National Policy on Traditional Medicine and Regulation of Herbal
Medicines explained that various types of traditional medicine (TM) and medical
practices referred to as complementary or alternative medicine (CAM) have been
increasingly used in both developing and developed countries. One of the major
components of the WHO Traditional Medicine Strategy is to promote the integration of
such medicine within a national health care system.
The use of medicinal plants is the most common form of traditional medication
worldwide. Regulation of herbal medicines is a key means of ensuring safety, efficacy
and quality of herbal medicinal products. WHO has been receiving an increasing number
of requests from governments for guidance on how to regulate herbal medicines.
During the last four years, many countries have established, or initiated the process
of establishing national regulations regarding herbal medicines. WHO has been
conducting a global survey on national policy on traditional medicine and on the
regulation of herbal medicines aiming to: (1) Collect updated and comprehensive
information on TM/CAM policies and regulations of herbal medicines; (2) Clarify the
current situation, in each country, on the TM/CAM policies and regulations of herbal
medicines, and their major challenges on these particular area; (3) Identify the specific
needs on capacity building for TM/CAM policy development including establishment of
regulations of herbal medicines, and the type of direct support WHO should provide to
member states; and (4) Monitor the impact of the WHO Strategy for Traditional
Medicine in relation to present national policy and regulation on TM/CAM/herbal
medicines.
WHO had received completed survey return from 141 countries. The raw data of
the survey results were fed into a database specifically designed for this project, to create
basic country profiles. Government clearance has been obtained for each country profile;
the manuscript of the draft summary report was finalized in English. The present
document provides a summary of the results of the WHO global survey with information
from 141 member states.
The baseline information gathered in the first of its kind, will be valuable not only
to help countries compare and learn each other’s experiences in strengthen their current
TM/CAM system, but also for guiding WHO on provision of support to member states.
National policy on traditional medicine and regulation of herbal medicines: Report
of a WHO global survey.
BIBLIOGRAPHY
Books
Cuevas, Frances Prescilla L., Editor in chief (2007). Public Health Nursing in the Philippines; Publication Committee, National League of Philippine
Government Nurses, Incorporated 11th Ed.
Govoni, Laura E. & Janice E. Hayes (2002). Drugs and Nursing Implications;Meredith Publishing Company.
Gutierez, Kathleen & Sherry F. Queener (2005). Pharmacology for Nursing; Mosby Inc.
Kozier, Barbara, et. Al.(2006). Fundamentals of Nursing: Concepts, Process, and Practice 8th ed. Pearson Education Inc.
Ramont, Robeta Pavy, Maldonado, Dolores and Towle, Mary Ann. (2006). Comprehensive Nursing Care. United States of America. Pearson Education, Inc..
Venzon, Lydia M. (2004). Introduction to Nursing Research; Quest for Quality Nursing. Quezon Avenue.
Wan et al. Biochemistry 47 2008
Electronic Sources: Internet
Fogoros,Richard N., M.D., (2003) – Retrieved from http://heartdisease.about.com/cs/-heartattacks/a/ clotting.htm
Hara, Hari C., (2005) – Retrieved from http://www.shvoong.com/exact-sciences/biology/1757270-blood-clotting-mechanism/
Janzen, (2005) – Retrieved from http://www.filipinoherbshealingwonders. filipinovegetarian-recipe.com/ balanoy.htm
Jesse Cris. (2008) – Retrieved from “The Feasibility of Using Mayana (Colleus scutellarioides) as Biological Stain”
Poller L. et.al.(1972) – Retrieved from http://www.ncbi.nlm.nih.gov/pmc/ articles/PMC477616/ “The Partial Thromboplastin (cephalin) Time Test
Ruggiero F M et. al. (1981)- Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/7259878 “Comparative Study of Lecithin and Cephalin Exchange between Liposomes and Mitochondria”
Samuel A. Guttman (1944)– Retrieved from http://www.ncbi.nlm.nih.gov/pmc/ articles/PMC435458/pdf/jcinvest00592-0046.pdf “Study on Cephalin-Cholesterol-Flocculation Reaction”
Wadsworth, Augustus et. al (1934) – Retrieved from http://www.jimmunol.org/ content/26/1/25.abstract “Study of the Antigenic Properties of Lecithin and Cephalin”
http://www.filipinoherbshealingwonders.filipinovegetarianrecipe.com/mayana.htm
http://www.herbanext.com/herbs-mayana
CHAPTER III
MATERIALS AND METHODS
This chapter will present the research methodology of the study. It includes the
research methodology, materials, equipment and apparatus, procedure training of the
panelists, evaluation of the products and statistical treatment of data.
Research Method
This study uses the descriptive-comparative as experimental design. It describes
the effectiveness of the crude extract of mayana leaves when used in blood coagulation
process. It also correlates the accurate blood clotting time using cephalin as positive
control, the crude extract of mayana leaves as the experimental specimen and the normal
blood clotting time as the negative control. The experimental research controls the
condition of the study. Parallel-group design is the type of experimental design used.
Wherein the normal coagulation time of blood serves as control in comparing the
effectiveness of the crude extract of mayana leaves with the effect of cephalin when used
by different blood types which are A, B, AB and O in coagulation process.
Materials
The materials to be used in this proposed study will be 70% alcohol, crude extract of
mayana, cephalin, A, B, AB, O blood types. Table 1 presents the materials used in the
extraction of crude extract from the mayana leaves (Coeus Blumei benth) and used in the
coagulation time.
Table 1
Materials Quantity
70% alcohol
crude extract of mayana
cephalin
Blood types:
A (3 samples)
B (3 samples)
AB (3 samples)
O (3 samples)
60ml
240μL
240μL
0.18cc
0.18cc
0.18cc
0.18cc
Table 1. Materials used in the extraction of crude extract from the mayana leaves (Coeus
Blumei benth) and used in the coagulation time.
Apparatus
The apparatuses to be used in this proposed study will be clean glass slides, cotton,
disposable lancet, needle, sterile gauze, stopwatch, mortar and pestle, beaker and knife.
Table 2 presents the apparatus used in the extraction of crude extract from the mayana
leaves (Coeus Blumei benth) and used in the coagulation time.
Table 2
Apparatus Quantity
Clean glass slides
Cotton
Disposable lancet (sterile)
Needle
Sterile gauze
Stopwatch
Mortar and pestle
Beaker
Knife
36 pieces
1 pack
36 pieces
36 pieces
1 piece
3 units
1 set
1 piece
1 piece
Table 2. Apparatus used in the extraction of crude extract from the mayana leaves (Coeus
Blumei benth) and used in the coagulation time.
Procedures
The procedure that will be used in conducting the study for coagulation time or
clotting time when the crude extract of mayana leaves is used in different blood types is
called the Slide Method or Drop Method. The steps or procedures in this study will be
done following in this order: (1) Disinfect site of puncture with 70% alcohol sponge and
dry. (2) Puncture to a depth of 3mm. (3) Wipe off first drop of blood. (4) Start the timer
as soon as the second drop of blood appears. (5) Transfer the second drop of blood onto
the center of glass slide. (6) Pass the tip of needle through the drop of blood every thirty
seconds and note for the formation of fibrin strands. (7) Stop the timer as soon as fibrin
strands are seen clinging at the tip of the needle.
The normal value of blood coagulation time in this test is said to be two to four (2-
4) minutes. After conducting the test to the four different blood types, the result will be
noted weather there are differences in the coagulation time of blood types A, B, AB and
O. The time noted will be used as a negative control on the next procedure where testing
the effectivity of mayana leaves in coagulation process while cephalin will be used as
positive control.
Repeat procedure one to five. (6) When the second drop of blood is onto the center
of the glass slide, get a 10μL of crude extract of mayana leaves and mix it with the
specimen. (7) Pass the tip of needle through the drop of blood with a crude extract every
thirty seconds and note for the formation of fibrin strands. (8) Stop the timer as soon as
fibrin strands are seen clinging at the tip of the needle. Then repeat the same procedure
for test of cephalin.
After noting all results in every procedures of this experiment. Tabulate the data
for better comparison. The researchers will write their conclusion on the effectiveness of
crude extract of mayana leaves in blood coagulation time based from the experiment they
conducted.
To obtain a crude extract from mayana leaves. The researchers will do these
following procedures. (1) Collect the mayana leaves early in the morning, before the heat
of the day. Choose mature leaves that are fully formed and mature but are not aged or
damaged. (2) Wash the mayana leaves with clean water. (3) Finely chop the leaves with
the knife to easily release all the components from the plant. (4) Pound the leaves using a
mortar and pestle to make extract. (5) Place the crude extract in the beaker. The container
should be cleaned, dried and sterilized with boiling water before use for a pure extract.
Figure 2.1
Crude extract of mayana leaves
Figure 2.1 Process flow sheet for the extraction of crude extract from the mayana leaves (Coeus Blumei benth) that will be used as experimental material for the study.
Wash the mayana leaves with clean water
Pound the leaves using a mortar and pestle to make extract.
Collect the mayana leaves early in the morning. Choose mature leaves that are fully formed.
Finely chop the leaves with the knife.
Place the crude extract in the beaker.
Figure 2.2
Blood Type A Blood Type B Blood Type AB Blood Type O
Figure 2.2 Process flow sheet for the comparison of the four different blood types (A, B, AB, O) as the crude extract of mayana leaves and cephalin are used in the coagulation time, comparing the result with the natural clotting time of the blood.
Natural Blood Clotting time
(- control)
Crude extract of mayana leaves(Experimental)
Cephalin(+ control)
Disinfect site of puncture with 70% alcohol sponge and dry
Puncture to a depth of 3mm
Wipe off first drop of blood
Start the timer as soon as the second drop of blood appears
Transfer the second drop of blood onto the center of glass slide
Pass the tip of needle through the drop of blood every thirty seconds and note
for the formation of fibrin strands
Stop the timer as soon as fibrin strands are seen clinging at the tip of the
needle
Conclusion
Get a 10μL of crude extract of mayana
leaves/cephalin and mix it with the specimen